Laser optical techniques and NMR relaxation methods are applied to study the dynamics of various Brownian motions, including twisting and bending deformations and conformational fluctuations, of DNA and other biophysically important macromolecules. Our goals are to ascertain the effects of various perturbations on the twisting and bending rigidities and, especially, to monitor long-range changes in secondary structure induced by sequence changes, supercoiling, bending strain, binding of regulatory proteins, and loss of bound water. Our main optical techniques are fluorescence polarization anisotropy (FPA), a new transient polarization grating (TPG) method, and coherent dynamic light scattering (DLS). The time-resolved FPA and TPG methods use polarized light pulses to photoselect those chromophores with the most favorable antenna orientations. The subsequent emission or absorption of light is then initially polarized, or anisotropic, but this anisotropy decays over time as a consequence of Brownian reorientations and can be monitored using fast photon detection and timing techniques. DLS uses cw laser light and digital autocorrelation of the scattered photons to monitor the dynamics of interference fluctuations produced by the randomly moving scattering elements in the sample.
The simplest Brownian motions accessible by these techniques are uniform end-over-end rotation, uniform spinning, and translational diffusion, which provide “hydrodynamic” information about molecular size and shape, including permanent bends. Deformational Brownian motions, such as bending, twisting, and distortion of the coil-envelopes of large DNAs, provide information about the mechanical properties of the DNA, which are remarkably sensitive to changes in secondary structure. Local group librations are also investigated by NMR relaxation methods.
The thermodynamics of supercoiled DNAs are investigated via equilibrium topoisomer distributions and the binding of unwinding ligands, while their structures are assessed by static and dynamic light scattering. Monte Carlo simulations of the thermodynamic and structural properties of supercoiled DNAs are performed to guide the interpretation of results. The effects of sequence changes and site-specific binding of regulatory proteins on the internal dynamics and structure of the DNA are studied by inserting specific sequences using molecular biology methods. The ongoing development of novel analytical theories and new algorithms for Monte Carlo and Brownian dynamics simulations requires techniques and concepts from a number of areas, such as thermodynamics, statistical mechanics, Gaussian random processes, hydrodynamics, and quantum mechanics.